Method for preventing hiv-1 infection of cd4+ cells

ABSTRACT

This invention provides methods for inhibiting fusion of HIV-1 to CD4 +  cells, comprising contacting CD4 +  cells with a non-chemokine agent capable of binding to a chemokine receptor in an amount and under conditions such that fusion of HIV-1 to CD4 +  cells is inhibited. Also provided are methods for inhibiting HIV-1 infection of CD4 +  cells, comprising contacting CD4 +  cells with a non-chemokine agent capable of binding to a chemokine receptor in an amount and under conditions such that fusion of HIV-1 to CD4 +  cells is inhibited, thereby inhibiting the HIV-1 infection. This invention provides non-chemokine agents capable of binding to the chemokine receptor and inhibiting fusion of HIV-1 to CD4 +  cells and pharmaceutical compositions comprising an amount of the non-chemokine agent capable of binding to the chemokine receptor and inhibiting fusion of HIV-1 to CD4 +  cells effective to prevent fusion of HIV-1 to CD4 +  cells and a pharmaceutically acceptable carrier.

This application is a continuation-in-part application of U.S. Ser. No.08/876,078, filed Jun. 13, 1997, the contents of which is herebyincorporated by reference.

Throughout this application, various references are referred to withinparentheses. Disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains. Fullbibliographic citation for these references may be found at the end ofeach series of experiments.

BACKGROUND OF THE INVENTION

Chemokines are a family of related soluble proteins of molecular weightbetween 8 and 10 KDa, secreted by lymphocytes and other cells, whichbind receptors on target cell surfaces resulting in the activation andmobilization of leukocytes, for example in the inflammatory process.Recently, Cocchi et al. demonstrated that the chemokines RANTES, MIP-1αand MIP-1β are factors produced by CD8⁺ T lymphocytes which inhibitinfection by macrophage-tropic primary isolates of HIV-1, but notinfection by laboratory-adapted strains of the virus (1). Thesechemokines are members of the C-C group of chemokines, so named becausethey have adjacent cysteine residues, unlike the C-X-C group which has asingle amino acid separating these residues (2). while Cocchi et al.found that expression of HIV-1 RNA was suppressed by treatment with thechemokines, they did not identify the site of action of these molecules.

A resonance energy transfer (RET) assay of HIV-1 envelopeglycoprotein-mediated membrane fusion was used to determine whetherfusion mediated by the envelope glycoprotein from the primarymacrophage-tropic isolate of HIV-1_(JR-FL) would be specificallyinhibited by chemokines, when compared with fusion mediated by theenvelope glycoprotein from the laboratory-adapted T lymphotrophic strainHIV-1_(LAI). As described below, it was demonstrated that this is indeedthe case. This demonstrates that some chemokine receptors are fusionaccessory molecules required for HIV-1 infection. Previous studies haveindicated that unidentified cell surface molecules are required forvirus entry in addition to the HIV-1 receptor, CD4. While CD4 isrequired for HIV-1 attachment, the accessory molecules are required forthe membrane fusion step of entry. These accessory molecules aregenerally expressed only on human cells, so HIV-1 does not infectnon-human CD4⁺ cells (3-6). Moreover it is possible to complementnon-human CD4⁺ cells by fusing them (using polyethylene glycol) withCD4⁻ human cells, resulting in a heterokaryon which is a competenttarget for HIV-1 envelope-mediated membrane fusion (7,8). These studieshave been performed using laboratory-adapted T lymphotrophic strains ofthe virus.

In some cases, it appears that fusion accessory molecules are found on asubset of human CD4⁺ cells and are required for infection by HIV-1isolates with particular tropisms. For example, macrophage-tropicprimary strains of HIV-1 such as HIv-1_(JR-FL) may have differentrequirements for accessory molecules compared with laboratory-adapted Tlymphotrophic strains such as HIV-1_(LAI). This phenomenon may explaindifferences in tropism between HIV-1 strains.

The current invention comprises a series of new therapeutics for HIV-1infection. It was demonstrated for the first time that chemokines act atthe fusion step of HIV-1 entry and specifically inhibit membrane fusionmediated by the envelope glycoprotein of primary macrophage-tropicprimary viral isolates, not laboratory-adapted T lymphotrophic strainsof the virus. Primary macrophage-tropic isolates of the virus are ofparticular importance since they are the strains usually involved invirus transmission, and may have particular importance in thepathogenesis of HIV-1 infection.

These results were obtained using a resonance energy transfer (RET)assay of HIV-1 envelope-mediated membrane fusion. Moreover, this assayis used to identify non-chemokines, including fragments of chemokinesand modified chemokines, that inhibit HIV-1 envelopeglycoprotein-mediated membrane fusion and thereby neutralize the virus,yet do not induce an inflammatory response.

SUMMARY OF THE INVENTION

This invention provides a method for inhibiting fusion of HIV-1 to CD4⁺cells which comprises contacting CD4 cells with a non-chemokine agentcapable of binding to a chemokine receptor in an amount and underconditions such that fusion of HIV-1 to the CD4⁺ cells is inhibited.

This invention also provides a method for inhibiting HIV-1 infection ofCD4⁺ cells which comprises contacting CD4⁺ cells with a non-chemokineagent capable of binding to a chemokine receptor in an amount and underconditions such that fusion of HIV-1 to the CD4⁺ cells is inhibited,thereby inhibiting the HIV-1 infection.

This invention further provides non-chemokine agents capable of bindingto the chemokine receptor and inhibiting fusion of HIV-1 to CD4⁺ cells.

This invention provides an agent which is capable of binding to fussingand inhibiting infection. In an embodiment, the agent is anoligopeptide. In another embodiment, the agent is an polypeptide. Instill another embodiment, the agent is an antibody or a portion of anantibody. In a separate embodiment, the agent is a nonypeptidyl agent.

In addition, this invention provides pharmaceutical compositionscomprising an amount of the above non-chemokine agents or agents capableof binding to fusin effective to inhibit fusion of HIV-1 to CD4⁺ cellsand a pharmaceutically acceptable carrier.

This invention provides a composition of matter capable of binding tothe chemokine receptor and inhibiting fusion of HIV-1 to CD4⁺ cellscomprising a non-chemokine agent linked to a ligand capable of bindingto a cell surface receptor of the CD4⁺ cells other than the chemokinereceptor such that the binding of the non-chemokine agent to thechemokine receptor does not prevent the binding of the ligand to theother receptor.

This invention also provides a pharmaceutical composition comprising anamount of the above-described composition of matter effective to inhibitfusion of HIV-1 to CD4⁺ cells and a pharmaceutically acceptable carrier.

This invention provides a composition of matter capable of binding tothe chemokine receptor and inhibiting fusion of HIV-1 to CD4⁺ cellscomprising a non-chemokine agent linked to a compound capable ofincreasing the in vivo half-life of the non-chemokine agent.

This invention also provides a pharmaceutical composition comprising anamount of a composition of matter comprising a non-chemokine agentlinked to a compound capable of increasing the in vivo half-life of thenon-chemokine agent effective to inhibit fusion of HIV-1 to CD4⁺ cellsand a pharmaceutically acceptable carrier.

This invention provide methods for reducing the likelihood of HIV-1infection in a subject comprising administering an above-describedpharmaceutical composition to the subject. This invention also providesmethods for treating HIV-1 infection in a subject comprisingadministering an above-described pharmaceutical composition to thesubject.

This invention also provides methods for determining whether anon-chemokine agent is capable of inhibiting the fusion of HIV-1 to aCD4⁺ cell which comprise: (a) contacting (i) a CD4⁺ cell which islabeled with a first dye and (ii) a cell expressing the HIV-1 envelopeglycoprotein on its surface which is labeled with a second dye, in thepresence of an excess of the agent under conditions permitting thefusion of the CD4⁺ cell to the cell expressing the HIV-1 envelopeglycoprotein on its surface in the absence of the agent, the first andsecond dyes being selected so as to allow resonance energy transferbetween the dyes; (b) exposing the product of step (a) to conditionswhich would result in resonance energy transfer if fusion has occurred;and (c) determining whether there is a reduction of resonance energytransfer, when compared with the resonance energy transfer in theabsence of the agent, a decrease in transfer indicating that the agentis capable of inhibiting fusion of HIV-1 to CD4⁺ cells.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1. Membrane fusion mediated by the HIV-1_(JR-FL) envelopeglycoprotein is inhibited by RANTES, MIP-1α and MIP-1β.

-   -   % RET resulting from the fusion of PM1 cells and        HeLa-env_(JR-FL) (▪) or HeLa-env_(LAI) (└) was measured in the        presence and absence of recombinant human chemokines at a range        of concentrations: RANTES (80-2.5 ng/ml), MIP-1α(400-12.5 ng/ml)        and MIP-1β (200-6.25 ng/ml), as indicated. Chemokines were added        simultaneously with the cells at the initiation of a four hour        incubation. Data are representative of more than three        independent experiments which were run in duplicate. The percent        inhibition of RET is defined as follows:

% Inhibition=100·[(Max RET−Min RET)−(Exp RET−Min RET)]/(Max RET−Min RET)

where Max RET is the % RET value obtained at four hours with HeLa-envcells and CD4-expressing cells in the absence of an inhibitory compound;Exp RET is the % RET value obtained for the same cell combination in thepresence of an inhibitory compound and Min RET is the background % RETvalue obtained using HeLa cells in place of HeLa envelope-expressingcells.

FIG. 2. CD4:HIV-1 gp120 binding in the presence of human chemokines.

-   -   The binding of soluble human CD4 to HIV-1_(LAI) and        HIV-1_(JR-FL) gp120 was determined in an ELISA assay in the        presence and absence of the monoclonal antibody OKT4A or        recombinant human chemokines at a range of concentrations,        identical to those used in the RET inhibition studies of FIG. 1:        OKT4A (62-0.3 nM), RANTES (10.3-0.3 nM), MIP-1α (53.3-2.9 nM),        and MIP-1β (25.6-0.8 nM). Inhibitors were added simultaneously        with biotinylated HIV-1 gp120 to soluble CD4 coated microtiter        plates (Dynatech Laboratories, Inc., Chantilly, Va.). Following        a two hour incubation at room temperature and extensive washing,        an incubation with streptavidin-horseradish peroxidase was        performed for one hour at room temperature. Following additional        washes, substrate was added and the OD at 492 nm determined in        an ELISA plate reader. Data are representative of two        independent experiments which were run in quadruplicate.

FIG. 3. Specificity, time course and stage of β-chemokine inhibition ofHIV-1 replication.

-   -   (a) PM1 cells (1×10⁶) were preincubated with        RANTES+MIP-1α+MIP-1β (R/Mα/Mβ; 100 ng/ml of each) for 24 h        (−24 h) or 2 h (−2 h), then washed twice with phosphate buffered        saline (PBS). HIV-1 (BaL env-complemented) virus (50 ng of p24;        see legend to Table 1) was added for 2 h, then the cells were        washed and incubated for 48 h before measurement of luciferase        activity in cell lysates as described previously (10, 11).        Alternatively, virus and R/Mα/Mβ were added simultaneously to        cells, and at the indicated time points (1 h, 3 h. etc) the        cells were washed twice in PBS, resuspended in culture medium        and incubated for 48 h prior to luciferase assay. Time 0        represents the positive control, to which no β-chemokines were        added. +2 h represents the mixture of virus with cells for 2 h        prior to washing twice in PBS, addition of R/Mα/Mβ and        continuation of the culture for a further 48 h before luciferase        assay.    -   (b) PM1 cells (1×10⁶) were infected with HIV-1 (500 pg p24)        grown in CEM cells (NL4/3; lanes 1-4) or macrophages (ADA; lanes        5-8), in the presence of 500 ng/ml of RANTES (lanes 1 and 5) or        MIP-1β (lanes 2 and 6), or with no β-chemokine (lanes 4 and 8).        Lanes 3 and 7 are negative controls (no virus). All viral stocks        used for the PCR assay were treated with DNAse for 30 min at 37°        C., and tested for DNA contamination before use. After 2 h. the        cells were washed and resuspended in medium containing the same        β-chemokines for a further 8 h. DNA was then extracted from        infected cells using a DNA/RNA isolation kit (US Biochemicals).        First round nested PCR was performed with primers: U3+,        5′-CAAGGCTACTTCCCTGATTGGCAGAACTACACACCAGG-3′ (SEQ ID NO:1)        preGag, 5′-AGCAAGCCGAGTCCTGCGTCGAGAG-3′ (SEQ ID NO:2) and the        second round with primers: LTR-test, 5′-GGGACTTTCCGCTGGGGACTTTC        3′ (SEQ ID NO:3) LRC2, 5′-CCTGTTCGGGCGCCACTGCTAGAGATTTTCCAC 3′        (SEQ ID NO:4) in a Perkin Elmer 2400 cycler with the following        amplification cycles: 94° C. for 5 min, 35 cycles of 94° C. for        30 s, 55° C. for 30 s, 72° C. for 30 s, 72° C. for 7 min. M        indicates 1 kb DNA ladder; 1, 10, 100, 1000 indicate number of        reference plasmid (pAD8) copies. The assay can detect 100 copies        of reverse transcripts.

FIG. 4: HIV-1 env-mediated membrane fusion of cells transientlyexpressing C-C CKR-5.

-   -   Membrane fusion mediated by β-chemokine receptors expressed in        HeLa cells was demonstrated as follows: Cells were transfected        with control plasmid pcDNA3.1 or plasmid pcDNA3.1-CKR constructs        using lipofectin (Gibco BRL). The pcDNA3.1 plasmid carries a        T7-polymerase promoter and transient expression of β-chemokine        receptors was boosted by infecting cells with 1×10⁷ pfu of        vaccinia encoding the T7-polymerase (vFT7.3) 4 h        post-lipofection (9). Cells were then cultured overnight in        R18-containing media and were tested for their ability to fuse        with HeLa-JR-FL cells (filled columns) or HeLa-BRU cells        (hatched column) in the RET assay. The % RET with control HeLa        cells was between 3% and 4% irrespective of the transfected        plasmid.

FIG. 5 Membrane fusion mediated by the HIV_(LAI) envelope glycoproteinis inhibited by SDF-1.

-   -   % RET resulting from the fusion of PM1 cells and        HeLa-env_(JR-FL) or HeLa-env_(LAI) cells (as indicated on the        graph) was measured in the presence of recombinant SDF-1α        (Gryphon Science, San Francisco) at the indicated        concentrations. Experimental method as described in the legend        to FIG. 1.

FIG. 6. Flow cytometric analysis of the binding of sCD4-gp120 complexesto (a) CCR5⁻ and (b) CCR5 L1.2 cells, a marine pre-B lymphoma line.

-   -   Cells are incubated for 15 min. with equimolar (−100 nM)        mixtures of sCD4 and biotinylated HIV-1_(JR-FL) gp120 and then        stained with a streptavidin-phycoerythrin conjugate, fixed with        2% paraformaldehyde, and analyzed by FACS. Cell number is        plotted on the y-axis.

FIG. 7. Inhibition of HIV-1 envelope-mediated cell fusion by thebicyclam JM3100, measured using the RET assay, with the cellcombinations indicated.

FIG. 6. Binding of CD4 and gp120 to CCR5.

-   -   Recombinant soluble CD4 (sCD4) and recombinant gp120 were added        in a range of concentrations either individually or as an        equimolar molecular complex to recombinant L1.2 cells that        express human CCR5 on their cell surface. The recombinant        proteins were biotinylated as indicated. Binding was detected by        adding a streptavidin-phycoerythrin conjugate and measuring the        fluorescence emission at 590 nm following excitation at 530 nm.        The following species were tested:    -   b-LAI:sCD4: complex formed between scD4 and biotinylated        HIV-1_(LAI), gp120    -   b-JR-FL:sCD4: complex formed between sCD4 and biotinylated        HIV-1_(JR-FL) gp120    -   b-sCD4 alone: biotinylated sCD4 added in the absence of gp120    -   b-JR-FL alone: biotinylated HIV-1_(JR-FL) gp120 added in the        absence of sCD4    -   These data demonstrate that complexation of soluble CD4 and        gp120 is necessary for CCR5 binding, as minimal binding is        observed for sCD4 or gp120 alone. The data further demonstrate        that binding is observed for sCD4-gp120 complexes when the gp120        is derived from macrophage-tropic (e.g. JR-FL) but not T        cell-tropic (e.g., LAI) strains of HIV, as expected from the        known relationship between HIV-1 tropism and co-receptor usage.        All data have been corrected for residual background binding to        nontransfected CCR5-L1.2 cells. To enhance chemokine receptor        expression, both transfected and parental L1.2 cells were        treated with sodium butyrate prior to assay (Wu at al., J. Exp.        Med. 185:1681)

FIG. 9. The CCR5 Binding assay identifies and determines the potency ofinhibitors of the gp120-CCR5 interaction.

-   -   HIV-1 Inhibitory monoclonal antibodies were added in a range of        concentrations to recombinant L1.2 cells that express human CCR5        on their cell surface and used to compete the binding of a        complex formed between sCD4 and biotinylated HIV-1_(JR-FL) gp        120, whose binding was detected using a        streptavidin-phycoerythrin conjugate. PA-8, -9, -10, -11 and -12        are Progenics' monoclonal antibodies that inhibit HIV-1 entry,        while 2D7 is a commercially available (Pharmingen, San Diego,        Calif.) ant-CCR5 monoclonal antibody that inhibits HIV-1 entry.        To enhance chemokine receptor expression, both transfected and        parental L1.2 cells were treated with sodium butyrate prior to        assay (Wu et al., J. Exp. Med. 185:1681).

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a method for inhibiting fusion of HIV-1 to CD4⁺cells which comprises contacting CD4 cells with a non-chemokine agentcapable of binding to a chemokine receptor in an amount and underconditions such that fusion of HIV-1 to the CD4⁺ cells is inhibited.

This invention also provides a method for inhibiting HIV-1 infection ofCD4⁺ cells which comprises contacting CD4⁺ cells with a non-chemokineagent capable of binding to a chemokine receptor in an amount and underconditions such that fusion of HIV-1 to the CD4⁺ cells is inhibited,thereby inhibiting the HIV-1 infection.

In this invention, a chemokine means RANTES, MIP-1-α, MIP-1-β or anotherchemokine which blocks HIV-1 infection. A chemokine receptor means areceptor capable of binding RANTES, MIP-1-α, MIP-1-β or anotherchemokine which blocks HIV-1 infection. Such chemokine receptor includesbut not limited to CCR5, CXCR4, CCR3 and CCR-2b.

Throughout this application, the receptor “fusin” is also named CXCR4and the chemokine receptor C-C CKR5 is also named CCR5.

The HIV-1 used in this application unless specified will mean clinicalor primary or field isolates or HIV-1 viruses which maintain theirclinical characteristics. The HIV-1 clinical isolates may be passaged inprimary peripheral blood mononuclear cells. The HIV-1 clinical isolatesmay be macrophage-trophic.

The non-chemokine agents of this invention are capable of binding tochemokine receptors and inhibiting fusion of HIV-1 to CD4⁺ cells. Thenon-chemokine agents include, but are not limited to, chemokinefragments and chemokine derivatives and analogues, but do not includenaturally occurring chemokines. The non-chemokine agents includemultimeric form a of the chemokine fragments and chemokine derivativesand analogues or fusion molecules which contain chemokine fragments,derivatives and analogues linked to other molecules.

The non-chemokine agents do not include bicyclams and their derivativesas described in U.S. Pat. No. 5,021,409, issued Jun. 4, 1991, thecontent of which is incorporated by reference into this application.Some bicyclam derivatives have been previously described with antiviralactivities (15, 16).

In an embodiment of this invention, the non-chemokine agent is anoligopeptide. In another embodiment, the non-chemokine agent is apolypeptide. In still another embodiment, the non-chemokine agent is anantibody or a portion thereof. Antibodies against the chemokine receptormay easily be generated by routine experiments. It is also within thelevel of ordinary skill to synthesize fragments of the antibody capableof binding to the chemokine receptor. In a further embodiment, thenon-chemokine agent is a nonpeptidyl agent.

Non-chemokine agents which are purely peptidyl in composition can beeither chemically synthesized by solid-phase methods (Merrifield, 1966)or produced using recombinant technology in either prokaryotic oreukaryotic systems. The synthetic and recombinant methods are well knownin the art.

Non-chemokine agents which contain biotin or other nonpeptidyl groupscan be prepared by chemical modification of synthetic or recombinantchemokines or non-chemokine agents. One chemical modification methodinvolves periodate oxidation of the 2-amino alcohol present onchemokines or non-chemokine agents possessing serine or threonine astheir N-terminal amino acid (Geophegan and Stroh, 1992). The resultingaldehyde group can be used to link peptidyl or non-peptidyl groups tothe oxidized chemokine or non-chemokine agent by reductive amination,hydrazine, or other chemistries well known to those skilled in the art.

As used herein, a N-terminus of a protein should mean the terminus ofthe protein after it has been processed. In case of a secretory proteinwhich contains a cleavable signal sequence, the N-terminus of asecretory protein should be the terminus after the cleavage of a signalpeptide.

This invention provides a method of identifying these non-chemokineagents. One way of identifying such agents, including non-peptidylagents, that bind to a chemokine receptor and inhibit fusion of HIV-1 toCD4⁺ cells is to use the following assay: 1) Incubate soluble CD4 withbiotinylated gp120 from HIV-1_(JR-FL) or HIV-1_(LAI); 2) Incubate thiscomplex with CCR5 or CXCR4-expressing cells (for HIV-1_(JR-FL) orHIV-1_(LAI) gp120s, respectively) that do not express CD4, in thepresence of absence of a candidate inhibitor; 3) Wash and then incubatewith streptavidin-phycoerythrin; and 4) Wash and then measure the amountof bound gp120 using a flow cytometer or fluorometer and calculate thedegree of inhibition of binding by the inhibitor.

Alternative methods to detect bound gp120 can also be used in place ofthe biotinylated gp120-streptavidin-phycoerythrin method describedabove. For example, peroxidase-conjugated gp120 could be used in placeof the biotinylated gp120 and binding detected using an appropriatecolorimetric substrate for peroxidase, with a spectrometric readout.

This invention further provides the non-chemokine agents identified bythe above methods.

This invention provides a non-chemokine agent capable of binding to thechemokine receptor and inhibiting fusion of HIV-1 to CD4⁺ cells with theproviso that the agent is not a known bicyclam or its known derivatives.In an embodiment, the non-chemokine is a polypeptide. In a furtherembodiment, this polypeptide is a fragment of the chemokine RANTES (Gonget al., 1996). In a still further embodiment, the polypeptide may alsocomprise the RANTES sequence with deletion of the N-terminal amino acidsof said sequence. The deletion may be the first eight N-terminal aminoacids of the RANTES sequence (SEQ ID NO:5).

In a separate embodiment, the polypeptide may comprise the MIP-1αsequence with deletion of the N-terminal amino acids of said sequence.The deletion may be the first seven, eight, nine or ten N-terminal aminoacids of the MIP-1β sequence.

In another embodiment of non-chemokine agent, the polypeptide comprisesthe MIP-1β sequence with the N-terminal sequence modified by addition ofan amino acid or oligopeptide. In a separate embodiment, the polypeptidecomprises the MIP-1β sequence with the N-terminal sequence modified byremoving the N-terminal alanine and replaced it by serine or threonineand additional amino acid or oligopeptide or nonpeptidyl moiety. In afurther embodiment, the additional amino acid is methionine.

As described Infra in the section of Experimental Details, a cofactorfor HIV-1 fusion and entry was identified and designated “fusin” (Fenget al., 1996). This invention provides an agent which is capable ofbinding to fusin and inhibiting infection. In an embodiment, the agentis an oligopeptide. In another embodiment, the agent is an polypeptide.

In a further embodiment, the polypeptide comprises SDF-1 with deletionof the N-terminal amino acids of said sequence. The deletion may be thefirst six, seven, eight, or nine N-terminal amino acids of the SDF-1sequence.

This invention also provides the above non-chemokine agent, wherein thepolypeptide comprises SDF-1 sequence with the N-terminal sequencemodified to produce antagonistic effect to SDF-1. One modification is toreplace the N-terminal glycine of SDF-1 by serine and derivatized withbiotin. Another modification is to replace the N-terminal glycine ofSDF-1 by serine and derivatized with methionine. A further modificationis to add the N-terminus of SDF-1 with a methionine before the terminalglycine.

In still another embodiment, the agent is an antibody or a portion of anantibody. In a separate embodiment, the agent is a nonpeptidyl agent.

The agents capable of binding to fusin may be identified by screeningdifferent compounds for their capability to bind to fusin in vitro.

A suitable method has been described by Fowlkes, at al. (1994),international application number: PCT/US94/03143, internationalpublication number: NO 94/23025, the content of which is incorporated byreference into this application. Briefly, yeast cells having a pheromonesystem are engineered to express a heterologous surrogate of a yeastpheromone system protein. The surrogate incorporates fusin and undersome conditions performs in the pheromone system of the yeast cell afunction naturally performed by the corresponding yeast pheromone systemprotein. Such yeast cells are also engineered to express a library ofpeptides whereby a yeast cell containing a peptide which binds fusinexhibits modulation of the interaction of surrogate yeast pheromonesystem protein with the yeast pheromone system and this modulation is aselectable or screenable event. Similar approaches may be used toidentify agents capable of binding to both fusin and the chemokinereceptor C-C CKR-5.

This invention also provides pharmaceutical compositions comprising anamount of such non-chemokine agents or agents capable of binding tofusin effective to inhibit fusion of HIV-1 to CD4⁺ cells and apharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers are well known to those skilled inthe art. Such pharmaceutically acceptable carriers may be aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, saline and buffered media.Parenteral vehicles include sodium chloride solution, Ringer's dextrose,dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, antimicrobials, antioxidants, chelating agents, inertgases and the like.

This invention provides a composition of matter capable of binding tothe chemokine receptor and inhibiting fusion of HIV-1 to CD4⁺ cellscomprising a non-chemokine agent linked to a ligand capable of bindingto a cell surface receptor of the CD4⁺ cells other than the chemokinereceptor such that the binding of the non-chemokine agent to thechemokine receptor does not prevent the binding of the ligand to theother receptor. In an embodiment, the cell surface receptor is CD4. Inanother embodiment, the ligand is an antibody or a portion of anantibody.

This invention also provides a pharmaceutical composition comprising anamount of an above-described composition of matter effective to inhibitfusion of HIV-1 to CD4⁺ cells and a pharmaceutically acceptable carrier.

This invention provides a composition of matter capable of binding tothe chemokine receptor and inhibiting fusion of HIV-1 to CD4⁺ cellscomprising a non-chemokine agent linked to a compound capable ofincreasing the in vivo half-life of the non-chemokine agent. In anembodiment, the compound is polyethylene glycol.

This invention also provides a pharmaceutical composition comprising anamount of a composition of matter comprising a non-chemokine agentlinked to a compound capable of increasing the in vive half-life of thenon-chemokine agent effective to inhibit fusion of HIV-1 to CD4⁺ cellsand a pharmaceutically acceptable carrier.

This invention provide methods for reducing likelihood of HIV-1infection in a subject comprising administering the above-describedpharmaceutical compositions to the subject. This invention also providesmethods for treating HIV-1 infection in a subject comprisingadministering the above-described pharmaceutical compositions to thesubject.

This invention also provides methods for determining whether anon-chemokine agent is capable of inhibiting the fusion of HIV-1 to aCD4⁺ cell which comprise: (a) contacting (i) a CD4⁺ cell which islabeled with a first dye and (ii) a cell expressing the HIV-1 envelopeglycoprotein on its surface which is labeled with a second dye, in thepresence of an excess of the agent under conditions permitting thefusion of the CD4⁺ cell to the cell expressing the HIV-1 envelopeglycoprotein on its surface in the absence of the agent, the first andsecond dyes being selected so as to allow resonance energy transferbetween the dyes; (b) exposing the product of step (a) to conditionswhich would result in resonance energy transfer if fusion has occurred;and (c) determining whether there is a reduction of resonance energytransfer, when compared with the resonance energy transfer in theabsence of the agent, a decrease in transfer indicating that the agentis capable of inhibiting fusion of HIV-1 to CD4⁺ cells.

HIV-1 only fuses with appropriate CD4⁺ cells. For example,laboratory-adapted T lymphotrophic HIV-1 strains fuse with most CD4⁺human cells. Clinical HIV-1 isolates do not fuse with most transformedCD4⁺ human cell lines but do fuse with human primary CD4⁺ cells such asCD4 T lymphocytes and macrophages. Routine experiments may be easilyperformed to determine whether the CD4⁺ cell is appropriate for theabove fusion assay.

As described in this invention, HIV-1 membrane fusion is monitored by aresonance energy transfer assay. The assay was described in theInternational Application Number, PCT/US94/14561, filed Dec. 16, 1994with International Publication Number WO 95/16789. This assay is furtherelaborated in a United States co-pending application Ser. No.08/475,515, filed Jun. 7, 1995. The contents of these applications arehereby incorporated by reference into this application.

In an embodiment of the above method, the non-chemokine agent is anoligopeptide. In another embodiment, the non-chemokine agent is apolypeptide. In still another embodiment, the agent is an antibody or aportion thereof. In a further embodiment, the non-chemokine agent is anonpeptidyl agent.

In a separate embodiment, the CD4⁺ cell is a PM1 cell. In anotherembodiment, the cell expressing the HIV-1 envelope glycoprotein is aHeLa cell expressing HIV-1_(JR-FL) gp120/gp41.

This invention provides a method for determining whether an agent iscapable of inhibiting HIV-1 infection comprising steps of: (a)contacting an appropriate concentration of an agent with a chemokinereceptor or a portion thereof under conditions permitting the binding ofthe agent to the chemokine receptor; (b) contacting the chemokinereceptor resulting from step (a) with a gp120/CD4 complex underconditions permitting the binding of the gp120/CD4 complex and thechemokine receptor in the absence of the agent; (c) measuring the amountof bound gp120/CD4 complex wherein a decrease in the amount comparedwith the amount determined in the absence of the agent indicates thatthe agent is capable of inhibiting HIV-1 infection.

As used herein, the portion of the chemokine receptor used in the abovemethod is the portion which maintains the capability of binding to HIV,i.e. capable of interaction with the gp120/CD4 complex. It is theintention of this invention to cover hybrid molecules or geneticallyengineered molecules which comprise this portion or domain of thechemokine receptor.

The gp120/CD4 complex used in the assay may include a truncated form ofeither molecules or hybrid proteins of molecules as long as the domainfor binding to the chemokine receptor is retained.

This invention provides a method for determining whether an agent iscapable of inhibiting HIV-1 infection comprising steps of: (a) fixing achemokine receptor on a solid matrix; (b) contacting the agent with thefixed chemokine receptor under conditions permitting the binding of theagent to the chemokine receptor; (c) removing the unbound agent; (d)contacting the fixed chemokine receptor resulting in step (c) with agp120 in the presence of CD4 under conditions permitting the binding ofthe gp120/CD4 complex and the chemokine receptor in the absence of theagent; (e) measuring the amount of bound gp120/CD4 complex; and (f)comparing the amount determined in step (d) with the amount determinedin the absence of the agent, a decrease of the amount indicating thatthe agent is capable of inhibiting HIV-1 infection.

This invention also provides a method for determining whether an agentis capable of inhibiting HIV-1 infection comprising steps of: (a) fixinga chemokine receptor on a solid matrix; (b) contacting the agent withthe fixed chemokine receptor; (c) contacting the mixture in step (b)with a gp120 in the presence of CD4 under conditions permitting thebinding of the gp120/CD4 complex and the chemokine receptor in theabsence of the agent; (d) measuring the amount of bound gp120/CD4complex; and (e) comparing the amount determined in step (d) with theamount determined in the absence of the agent, a decrease of the amountindicating that the agent is capable of inhibiting HIV-1 infection.

This invention also provides a method for determining whether an agentis capable of inhibiting HIV-1 infection comprising steps of: (a)contacting the agent with a gp120/CD4 complex under conditionspermitting the binding of the agent to the gp120/CD4 complex; (b)contacting the gp120/CD4 complex resulting from step (a) with achemokine receptor under conditions permitting the binding of thegp120/CD4 complex and the chemokine receptor in the absence of theagent; (c) measuring the amount of bound chemokine receptor, wherein adecrease of the amount when compared with the amount determined in theabsence of the agent indicates that the agent is capable of inhibitingHIV-1 infection.

This invention also provides a method for determining whether an agentis capable of inhibiting HIV-1 infection comprising steps of: (a) fixinga gp120/CD4 complex on a solid matrix; (b) contacting the agent with thefixed gp120/CD4 complex under conditions permitting the binding of theagent to the gp120/CD4 complex; (c) removing unbound agent; (d)contacting the fixed gp120/CD4 complex resulting from step (c) with achemokine receptor under conditions permitting the binding of thegp120/CD4 complex and the chemokine receptor in the absence of theagent; (e) measuring the amount of bound chemokine receptor; and (f)comparing the amount determined in step (a) with the amount determinedin the absence of the agent, a decrease of the amount indicating thatthe agent is capable of inhibiting HIV-1 infection.

This invention provides a method for determining whether an agent iscapable of inhibiting HIV-1 infection comprising steps of: (a) fixing agp120/CD4 on a solid matrix; (b) contacting the agent with the fixedgp120/CD4 complex; (c) contacting the mixture in step (b) with achemokine receptor under conditions permitting the binding of thegp120/CD4 complex and the chemokine receptor in the absence of theagent: (d) measuring the amount of bound chemokine receptor; (e)comparing the amount determined in step (d) with the amount determinedin the absence of the agent, a decrease of the amount indicating thatthe agent is capable of inhibiting HIV-1 infection.

As used in these assays, CD4 include soluble CD4, fragments of CD4 orpolypeptides incorporating the gp120 binding site of CD4 capable ofbinding gp120 and enabling the binding of gp120 to the appropriatechemokine receptor.

As used in these assay gp120 is the gp120 from an appropriate strain ofHIV-1. For example, gp120 from the macrophage tropic clinical isolateHIV-1_(JR-FL) will bind to the chemokine receptor CCR5, whereas gp120from the laboratory adapted T-tropic strain HIV-1_(LAI) will bind to thechemokine receptor CXCR4.

In a preferred embodiment of the above methods, the CD4 is a solubleCD4. The chemokine receptor which may be used in the above assayincludes CCR5, CXCR4, CCR3 and CCR-2b.

In an embodiment, the chemokine receptor is expressed on a cell. Inanother embodiment, the chemokine receptor is embedded in liposomes. Infurther embodiment, the chemokine receptor is embedded in a membranederived from cells expressing the chemokine receptor. In a preferredembodiment, the cell is a L1.2 cell. In a separate embodiment, thechemokine receptor is purified and reconstituted in liposomes. Suchchemokine receptor embedded in the lipid bilayer of liposomes retainsthe gp120 binding activity of the receptor.

The gp120, CD4 or both may be labelled with a detectable marker in theabove assays. Markers including radioisotope or enzymes such as horseradish peroxidase may be used in this invention.

In an embodiment, the gp120 or CD4 or the chemokine receptor is labelledwith biotin. In a further embodiment, the biotinylated gp120, or CD4 orthe chemokine receptor is detected by: (i) incubating withstreptavidin-phycoerythrin, (ii) washing the incubated mixture resultingfrom step (i), and (iii) measuring the amount of bound gp120 using aplate reader, exciting at 530 nm, reading emission at 590 nm.

This invention also provides an agent determined to be capable ofinhibiting HIV-1 infection by the above methods, which is previouslyunknown.

This invention also provides a pharmaceutical composition comprising theagent determined to be capable of inhibiting HIV-1 infection by theabove methods and a pharmaceutically acceptable carrier. In anembodiment, the agent is an oligopeptide. In another embodiment, theagent is a polypeptide. In a still another embodiment, the agent is anonpeptidyl agent.

This invention also provides a molecule capable of binding to thechemokine receptor CCR5 and inhibiting fusion of HIV-1 to CD4⁺ cellscomprising the above determined agent linked to a compound capable ofincreasing the in vivo half-life of the non-chemokine agent. In anembodiment, the compound is polyethylene glycol. This invention alsoprovides a pharmaceutical composition comprising an amount of the abovemolecule effective to inhibit HIV-1 infection and a pharmaceuticallyacceptable carrier.

This invention provides a method for reducing the likelihood of HIV-1infection in a subject comprising administering the above pharmaceuticalcompositions to the subject.

This invention provides a method for treating HIV-1 infection in asubject comprising administering the above pharmaceutical composition tothe subject.

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

EXPERIMENTAL DETAILS First Series of Experiments

1) Chemokines Inhibit Fusion Mediated by the Envelope Glycoprotein froma Macrophage-Tropic Primary Isolate of HIV-1 but not from aLaboratory-Adapted T-Lymphotrophic Strain of the Virus

The Chemokines RANTES, MIP-1α and MIP-1β were obtained from R & Dsystems (Minneapolis, Minn.). They were tested in the RET assay forability to inhibit fusion between HeLa-env_(JR-FL) cells (expressinggp120/gp41 from the macrophage tropic isolate HIV-1_(JR-FL)) and PM1cells, or for inhibition of fusion between HeLa-env_(LAI) cells(expressing gp120/gp41 from the laboratory-adapted strain HIV-1_(LAI))and various CD4 T lymphocyte cell lines. As shown in FIG. 1, all threechemokines inhibited fusion mediated by the macrophage tropic virusenvelope glycoprotein, but not that mediated by the laboratory-adaptedstrain envelope glycoprotein.

The ability of the chemokines to block the interaction between CD4 andHIV-1 gp120 which occurs at virus attachment was then tested. It wasfound that the chemokines did not inhibit this interaction (FIG. 2),demonstrating that their blockade of HIV-1 envelopeglycoprotein-mediated membrane fusion occurs at the membrane fusionevent itself, rather than the initial CD4-gp120 interaction whichprecedes fusion.

2) Non-Chemokine Peptides and Derivatives that Inhibit HIV-1 Fusion

The non-chemokines include chemokine fragments and chemokine derivativesthat are tested in the RET assay to determine which are active ininhibiting HIV-1 membrane fusion. Particular attention is focused onfragments or derivatives that inhibit HIV-1 fusion but do not activateleukocyte responses. These non-chemokines include:

a) N-terminal derivatives of the chemokines. Addition of residues to theN-terminus of chemokines inhibits the function of these proteins withoutsignificantly reducing their ability to bind chemokine receptors. Forexample, Met-RANTES (RANTES with an N-terminal methionine) has beenshown to be a powerful antagonist of native RANTES and is unable toinduce chemotaxis or calcium mobilization in certain systems. Themechanism of antagonism appears to be competition for receptor binding(9). Similar results were found using other derivatives of the Nterminus of RANTES (9) and also by N-terminal modification of otherchemokines, such as IL-8 (a member of the C-X-C chemokines) (10). Thecurrent invention includes Met-RANTES and other chemokines derivatisedby the addition of methionine, or other residues, to the N-terminus sothat they inhibit fusion mediated by the envelope glycoprotein ofHIV-1_(JR-FL), and inhibit infection by many isolates of HIV-1, yet donot activate the inflammatory response.b) Chemokines with N-terminal amino acids deleted: Chemokine antagonistshave been generated by deleting amino acids in the N-terminal region.For example, deletion of up to 8 amino acids at the N-terminus of thechemokine MCP-1 (a member of the C-C chemokine group), ablated thebioactivity of the protein while allowing it to retain chemokinereceptor binding and the ability to inhibit activity of native MCP-1(11,12).

The current invention includes N-terminal deletants of RANTES, MIP-1αand MIP-1β, lacking the biological activity of the native proteins,which inhibit HIV-1 fusion and HIV-1 infection.

c) Other peptides: A series of overlapping peptides (e.g. of 20-67residues) from all regions of RANTES, MIP-1α and MIP-1β are screened bythe same approaches to identify peptides which inhibit HIV-1 fusion mostpotently without activating leukocytes. Activation of leukocyteresponses is measured following routine procedures (9, 10, 11, 12).

3) Cloning the Chemokine Receptors

Chemokine receptors required for HIV-1 fusion are cloned by thefollowing strategy. First a cDNA library is made in a mammalianexpression vector (e.g. pcDNA3.1 from Invitrogen Corp. San Diego,Calif.) using mRNA prepared from the PM1 cell line or CD4⁺ T-lymphocytesor macrophages. Degenerate oligonucleotide probes are used to identifymembers of the cDNA library encoding members of the chemokine receptorfamily, for example following previously published methods (2). Thevectors containing chemokine receptor cDNAs are then individuallyexpressed in one of several mammalian cell lines which express human CD4but do not fuse with HeLa-env_(JR-FL) cells (e.g. HeLa-CD4, CHO-CD4 orCOS-CD4) or HeLa-env_(LAI) cells (e.g. CHO-CD4 or COS-CD4). Followinganalysis in the RET assay, clones which gain the ability to fuse withHeLa-env_(JR-FL) or HeLa-env_(LAI) are identified and the codingsequences recovered, for example by PCR amplification, followingprocedures well known to those skilled in the art. DNA sequencing isthen performed to determine whether the cDNA recovered encodes a knownchemokine receptor. Following expression of the receptor, monoclonal andpolyclonal antibodies are prepared and tested for ability to inhibitinfection by a panel of HIV-1 isolates.

REFERENCES OF THE FIRST SERIES OF EXPERIMENTS

-   1. Cocchi, F., DeVico, A. L., Garzino-Demo, A., Arya, S. K.,    Gallo, R. C., Lusso, P. 1995. Science. 270:1811-1815.-   2. Raport, C. J., Schweickart, V. L. Chantry, D., Eddy Jr., R. L.,    Shows, T. B., Godiska, R., Gray, P. W. 1996. Journal of Leukocyte    Biology. 59: 18-23.-   3. Maddon P J., Dalgleish A G., McDougal J S., Clapham P R., Weiss R    A., Axel R. 1986. Cell. 47:333-348.-   4. Ashorn P A., Berger E A., Moss B. 1990. J. Virol. 64:2149-2156.-   5. Clapham P R., Blanc D., Weiss R A. 1991. Virology. 181:703-715.-   6. Harrington R D., Geballe A P. 1993. J. Virol. 67:5939-5947.-   7. Broder C C., Dimitrov D S., Blumenthal R., Berger E A. 1993.    Virology. 193:483-491.-   8. Dragic T., Charneau P., Clavel P., Alizon M. 1992. J. Virol.    66:4794-4802.-   9. Wells, T. N., Power. C. A. Lusti-Narasimhan. M., Hoogewerf, A.    J., Cooke, R. M., Chung, C. W., Peitach, M. C., Proudfoot, A. E.    1996. Journal of Leukocyte Biology. 59:53-60.-   10. Moser, B., Dewald B., Barella, L., Schumacher, C., Baggiolini,    M., Clark-Lewis. I. 1993. Journal of Biological Chemistry.    268:7125-7128.-   11. Gong, J. H., Clark-Lewis. I. 1995. J. Exp. Med. 181:631-640.-   12. Zhang, Y. J., Rutledge. B. J., Rollins, B. 1994. Journal of    Biological Chemistry. 269:15918-15924.-   13. Merrifield, R. B. (1963) J. Am. Chem. Soc. 85: 2149-2154.-   14. Goeghegan, K. P. Stroh, J. P. (1992) Bioconjugate Chem. 3:    138-146.-   15. Clercq, E. D. et al. (1994) Antimicrobial Agents and    Chemotherapy 38:668-674.-   16. Clercq, E. D. et al (1992) Proc. Natl. Acad. Sci. USA 89:    5286-5290.

Second Series of Experiments

The replication of primary, non-syncytium-inducing (NSI) HIV-1 isolatesin CD4⁺ T-cells is inhibited by the C-C β-chemokines MIP-1α, MIP-1β andRANTES (1,2), but T-cell line-adapted (TCLA) or syncytium-inducing (SI)primary strains are insensitive (2,3). The β-chemokines are small (8kDa), related proteins active on cells of the lymphoid and monocytelineage (4-8). Their receptors are members of the 7-membrane-spanning,G-protein-linked superfamily, one of which (the LESTR orphan receptor)has been identified as the second receptor for TCLA HIV-1 strains, andis now designated fusin (9). Fusin is not known to be a β-chemokinereceptor (7-9).

To study how β-chemokines inhibit HIV-1 replication, a virus entry assaybased on single-cycle infection by an env-deficient virus, NL4/3Δenv(which also carries the luciferase reporter gene), complemented byenvelope glycoproteins expressed in trans was used (10,11). Variousenv-complemented viruses were tested in PM1 cells, a variant of HUT-78that has the unique ability to support replication of primary and TCLAHIV-1 strains, allowing comparison of envelope glycoprotein functionsagainst a common cellular background (2,12). MIP-1α, MIP-1β and RANTEare most active against HIV-1 in combination (2,3), and stronglyinhibited infection of PM1 cells by complemented viruses whose envelopesare derived from the NSI primary strains ADA and BaL (Table 1a).

TABLE 1 Inhibition of HIV-1 entry in PM1 cells and CD4⁺ T-cells byβ-chemokines % luciferase activity a) PM1 cells BaL ADA NL4/3 HxB2 MuLVcontrol without virus 2 2 2 5 3 control with virus 100 100 100 100 100+R/Mα/Mβ (50/50/50) 2 3 92 117 100 +RANTES (100) 1 1 nd nd nd +MIP-1α(100) 54 54 nd nd nd +MIP-1β (100) 1 6 nd nd nd +MCP-1 (100) 46 50 nd ndnd +MCP-2 (100) 28 26 nd nd nd +MCP-3 (100) 58 46 nd nd nd b) JR-FL HxB2MuLV LW4 CD4⁺ T-cells control without virus 1 1 1 control with virus 100100 100 +R/Mα/Mβ (200/200/200) 14 68 nd LW5 CD4⁺ T-cells control withoutvirus 1 1 1 control with virus 100 100 100 +R/Mα/Mβ (200/200/200) 15 73nd

Table 1 Legend:

P1 cells were cultured as described by Lusso et al (12).Ficoll/hypaque-solaced PBNC from laboratory workers (LW) stimulated withPHA for 72 h before depletion of CD8+ Lymphocyces with anti-CD8inmunamagnetic beads (DYNAL, Great Neck, N.Y.). CD4+ Lymphocytes weremaintained in culture medium containing interleukin-2 (100 U/ml; HofmannLaRoche, Nutley, N.J.), as described previously (3). Target cells(1-2×10⁵) were infected with supernatants (10-50 ng of HIV-1 p24) from293-cells co-transfected with an NL4/3Δenv-luciferase vector and a HIV-1env-expressing vector (10, 11). β-Chemokines (R & D systems,Minneapolis) were added to the target cells simultaneously with virus,at the final concentrations (ng/ml) indicated in parentheses in thefirst column. The β-chemokine concentration range was selected based onprior studies (2,3). After 2 h, the cells were washed twice with PBS,resuspended in β-chemokine-containing media and maintained for 48-96 h.Luciferase activity in cell lysates was measured as described previously(10,11). The values indicated represent luciferase activity (cpm)/ngp24/mg protein, expressed relative to that in virus-control cultureslacking β-chemokines (100%), and are the means of duplicate orsextuplicate determinations. nd, not done. R/Mα/Mβ,RANTES+MIP-1α+MIP-1β.

RANTES and MIP-1β were strongly active when added individually, whileother β-chemokines—MIP-1α, MCP-1, MCP-2 and MCP-3 (refs. 13-15)—wereweaker inhibitors (Table 1a). However, MIP-1α, MIP-1β and RANTES, incombination, did not inhibit infection of PM1 cells by the TCLA strainsNL4/3 and HxB2, or by the amphotropic murine leukemia virus (MuLV-Ampho)pseudotype (Table 1a). Thus, phenotypic characteristics of the HIV-1envelope glycoproteins influence their sensitivity to β-chemokines in avirus entry assay.

The env-complementation assay was used to assess HIV-1 entry into CD4+T-cells from two control individuals (LW4 and LW5). MIP-1α, MIP-1β andRANTES strongly inhibited infection by the NSI primary strain JR-FLinfection of LW4's and LW5's CD4⁺ T-cells, and weakly reduced HxB2infection of LW cells (Table 1b), suggesting that there may be someoverlap in receptor usage on activated CD4⁻ T-cells by different virusstrains. BaL env-mediated replication in normal PBL was also inhibitedby MIP-1α, MIP-1β and RANTES, albeit with significant inter-donorvariation in sensitivity (data not shown).

It was determined when β-chemokines inhibited HIV-1 replication byshowing that complete inhibition of infection of PM1 cells required thecontinuous presence of β-chemokines for up to 5 h after addition of ADAor BaL env-complemented virus (FIG. 3 a). Pre-treatment of the cellswith β-chemokines for 2 h or 24 h prior to infection had no inhibitoryeffect if the cells were subsequently washed before virus addition.Furthermore, adding β-chemokines 2 h after virus only minimally affectedvirus entry (FIG. 3 a). A PCR-based assay was next used to detect HIV-1early DNA reverse transcripts in PM1 cells after 10 h of infection;reverse transcription of ADA, but not of NL4/3, could not be detected inthe presence of MIP-1β and RANTES (FIG. 3 b). Thus, inhibition byβ-chemokines requires their presence during at least one of the earlystages of HIV-1 replication: virus attachment, fusion and early reversetranscription.

As described in part in the First Series of Experiments, these sites ofaction were discriminated, first by testing whether β-chemokinesinhibited binding of JR-FL or BRU (LAI) gp120 to soluble CD4, or oftetrameric CD4-IgG2 binding to HeLa-JR-FL cells expressing oligomericenvelope glycoproteins (17). No inhibition by any of the β-chemokineswas found in either assay, whereas the OKT4a CD4-MAb was stronglyinhibitory in both (FIG. 2 and data not shown). Thus, β-chemokinesinhibit a step after CD4 binding, when conformational changes in theenvelope glycoproteins lead to fusion of the viral and cellularmembranes (18). Cell-cell membrane fusion is also induced by thegp120-CD4 interaction, and can be monitored directly by resonance energytransfer (RET) between fluorescent dyes incorporated into cell membranes(17). In the RET assay, OKT4a completely inhibits membrane fusion of PM1cells with HeLa cells expressing the envelope glycoproteins of eitherJR-FL (HeLa-JR-FL, the same cell line referred to above asHeLa-env_(JR-FL)) or BRU (HeLa-BRU, the same cell line referred to aboveas HeLa-env_(LAI)), confirming the specificity of the process (17).RANTES, MIP-13 (and to a lesser extent, MIP-1α) strongly inhibitedmembrane fusion of HeLa-JR-FL cells with PM1 cells, whereas fusionbetween PM1 cells and HeLa-BRU cells was insensitive to theseβ-chemokines (FIG. 1 and Table 2a).

TABLE 2 Effect of β-chemokines on HIV-1 envelope glycoprotein- mediatedmembrane fusion measured using the RET assay % Fusion HeLa-JR-FLHeLa-BRU a) PM1 cells no chemokines 100 100 +R/Mα/Mβ (80/400/100) 1 95+RANTES (80) 8 100 +MIP-1α (400) 39 100 +MIP-1β (100) 13 93 +MCP-1 (100)99 98 +MCP-2 (100) 72 93 +MCP-3 (100) 98 99 b) LW5 CD4⁺ cells nochemokines 100 100 +R/Mα/Mβ(106/533/133) 39 100 +RANTES (106) 65 95+MIP-1α (533) 72 100 +MIP-1β (133) 44 92 +OKT4A (3 ug/ml) 0 0

Table 2 Legend:

CD4⁺ target cells (mitogen-activated CD4⁺ lymphocytes or PM1 cells) werelabeled with octadecyl rhodamine (Molecular Probes, Eugene, Oreg.), andHeLa-JR-FL cells, HeLa-BRU cells (or control HeLa cells, not shown) werelabeled with octadecyl fluorescein (Molecular Probes), overnight at37-C. Equal numbers of labeled target cells and env-expressing cellswere mixed in 96-well plates and β-chemokines for CD4 MAb OT4a) wereadded at the final concentrations (ng/ml) indicated in parentheses inthe first column. Fluorescence emission values were determined 4 h aftercell mixing (17). If cell fusion occurs, the dyes are closely associatedin the conjoined membrane such that excitation of fluorescein at 450 nmresults in resonance energy transfer (RET) and emission by rhodamine at590 nm. Percentage fusion is defined as equal to 100×[(Exp RET−MinRET)/(Max RET−Min RET)], where Max RET=% RET obtained when HeLa-Env andCD4⁺ cells are mixed, Exp RET=% RET obtained when HeLa-Env and CD4⁺cells are mixed in the presence of fusion-inhibitory compounds, and MinRET=% RET obtained when HeLa cells (lacking HIV-1 envelopeglycoproceins) and CD4⁺ cells are mixed. The % RET value is defined by acalculation described elsewhere (17), and each is the mean of triplicatedeterminations. These values were, for HeLa-JR-FL and HeLa-BRU cellsrespectively: PM1 cells 11.5%, 10.5%; LW5 CD4⁺ cells, 6.0%, 10.5%;R/Mα/Mβ, RANTES+MIP-1α+MIP-1β.

Similar results were obtained with primary CD4⁺ T-cells from LW5 (Table2b), although higher concentrations of β-chemokines were required toinhibit membrane fusion in the primary cells than in PM1 cells. Thus,the actions of the β-chemokines are not restricted to the PM1 cell line.The RET assay demonstrates that β-chemokines interfere with env-mediatedmembrane fusion.

The simplest explanation of these results is that the binding of certainβ-chemokines to their receptor(s) prevents, directly or otherwise, thefusion of HIV-1 with CD4⁺ T-cells. It has been known for a decade thatHIV-1 requires a second receptor for entry into CD4⁺ cells (19-21). Thisfunction is supplied, for TCLA strains, by fusin (9). Several receptorsfor MIP-1α, MIP-1β and RANTES have been identified (6.7), andβ-chemokines exhibit considerable cross-reactivity in receptor usage(4-8). However, C-C CKR-1 and, especially, C-C CKR-5 were identified asthe most likely candidates, based on tissue expression patterns andtheir abilities to bind MIP-1α, MIP-1β and RANTES (4, 7, 8, 15, 22). C-CCKR-1, C-C CKR-5 and LESTR are each expressed at the mRNA level in PM1cells and primary macrophages (data not shown). These and otherβ-chemokine receptors were therefore PCR-amplified, cloned andexpressed.

The expression of C-C CKR-5 in HeLa-CD4 (human). COS-CD4 (simian) and3T3-CD4 (murine) cells rendered each of them readily infectible by theprimary, NSI strains ADA and BaL in the env-complementation assay ofHIV-1 entry (Table 3)

TABLE 3 C-C CKR-5 expression permits infection of CD4-expressing cellsby primary, NSI HIV-1 strains R/Mα/Mβ pcDNA3.1 LESTR CKR-1 CKR-2a CKR-3CKR-4 CKR-5 CKR-5 COS-CD4 ADA 798 456 600 816 516 534 153000 3210 BaL660 378 600 636 516 618 58800 756 HxB2 5800 96700 5240 5070 5470 56204850 5000 HeLa-CD4 ADA 678 558 4500 912 558 600 310000 6336 BaL 630 7381800 654 516 636 104000 750 HxB2 337000 nd nd nd nd nd nd 356000 3T3-CD4ADA 468 558 450 618 534 606 28400 1220 BaL 606 738 660 738 534 558 11700756 HxB2 456 24800 618 672 732 606 618 606

Table 3 Legend:

Chemokine receptor genes C-C CKR-1, C-C CKR-2a, C-C CKR-3, C-C CKR-4 andC-C CKR-5 have no introns (4-8, 15, 22) and were isolated by PCRperformed directly on a human genomic DNA pool derived from the PBNC ofseven healthy donors. Oligonucleotides overlapping the ATG and the stopcodons and containing BamHI and Xhol restriction sites for directionalcloning into the pcDNA3.1 expression vector (Invitrogen Inc.) were used.LESTR (also known as fusin or HUMSTR) (4, 9, 24) was cloned by PCRperformed directly on cDNA derived from PM1 cells, using sequencesderived from the NIH database. Listed below are the 5′ and 3′ primerpairs used in first (5-1 and 3-1) and second (5-2 and 3-2) round PCRamplification of the CKR genes directly from human genomic DNA, and ofLESTR from PM1 cDNA. Only a single set of primers was used to amplifyCKR-5.

LESTR: (SEQ ID NO: 6) L/5-1 = AAG CTT GGA GAA CCA GCG GTT ACC ATG GAGGGG ATC; (SEQ ID NO: 7) L/5-2 = GTC TGA GTC TGA GTC AAG CTT GGA GAA CCA;(SEQ ID NO: 8) L/3-1 = CTC GAG CAT CTG TGT TAG CTG GAG TGA AAACTT GAA CAC TC; (SEQ ID NO: 9) L/3-2 =GTC TGA GTC TGA GTC CTC GAG CAT CTG TGT; CKR-1: (SEQ ID NO: 10) C1/5-1 =AAG CTT CAG AGA GAA GCC GGG ATG GAA ACT CC; (SEQ ID NO: 11) C1/5-2 =GTC TGA GTC TGA GTC AAG CTT CAG AGA GAA; (SEQ ID NO: 12) C1/3-1 =CTC GAG CTG AGT CAG AAC CCA GCA GAG AGT TC; (SEQ ID NO: 13) C1/3-2 =GTC TGA GTC TGA GTC CTC GAG CTG AGT CAG; CKR-2a: (SEQ ID NO: 14)C2/5-1 = AAG CTT CAG TAC ATC CAC AAC ATG CTG TCC AC; (SEQ ID NO: 15)C2/5-2 = GTC TGA GTC TGA GTC AAG CTT CAG TAC ATC; (SEQ ID NO: 16)C2/3-1 = CTC GAG CCT CGT TTT ATA AAC CAG CCG AGA C; (SEQ ID NO: 17)C2/3-2 = GTC TGA GTC TGA GTC CTC GAG CCT CGT TTT; CKR-3: (SEQ ID NO: 18)C3/5-1 = AAG CTT CAG GGA GAA GTG AAA TGA CAA CC; (SEQ ID NO: 19)C3/5-2 = GTC TGA GTC TGA GTC AAG CTT CAG GGA GAA; (SEQ ID NO: 20)C3/3-1 = CTC GAG CAG ACC TAA AAC ACA ATA GAG AGT TCC; (SEQ ID NO: 21)C3/3-2 = GTC TGA GTC TGA GTC CTC GAG CAG ACC TAA; CKR-4: (SEQ ID NO: 22)C4/5-1 = AAG CTT CTG TAG AGT TAA AAA ATG AAC CCC ACG G; (SEQ ID NO: 23)C4/5-2 = GTC TGA GTC TGA GTC AAG CTT CTG TAG AGT; (SEQ ID NO: 24)C4/3-1 = CTC GAG CCA TTT CAT TTT TCT ACA GGA CAG CAT C; (SEQ ID NO: 25)C4/3-2 = GTC TGA GTC TGA GTC CTC GAG CCA TTT CAT; CKR-5: (SEQ ID NO: 26)C5/5-12 = GTC TGA GTC TGA GTC AAG CTT AAC AAG ATG GAT TAT CAA;(SEQ ID NO: 37) C5/3-12 = GTC TGA GTC TGA GTC CTC GAG TCC GTG TCACAA GCC CAC.

The human CD4-expressing cell lines HeLa-CD4 (P42), 3T3-CD4 (sc6) andCOS-CD4 (Z28T1) (23) were transfected with the different pcDMA3.1-CKRconstructs by the calcium phosphate method, then infected 48 h laterwith different reporter viruses (200 ng of HIV-1 p24/10⁶ cells) in thepresence or absence of β-chemokines (400 ng/ml each of RANTES, MIP-1αand MIP-1β). Luciferase activity in cell lysates was measured 48 h later(10.11). β-Chemokine blocking data is only shown for C-C CKR-5, asinfection mediated by the other C-C CKR genes was too weak forinhibition to be quantifiable. In PCR-based assays of HIV-1 entry, a lowlevel of entry of NL4/3 and ADA into C-C CKR-1 expressing cells (datanot shown) was consistently observed.

Neither LESTR nor C-C CKR-1, -2a, -3 or -4 could substitute for C-CCKR-5 in this assay. The expression of LESTR in COS-CD4 and 3T3-CD4cells permitted HxB2 entry, and HxB2 readily entered untransfected (orcontrol plasmid-transfected) HeLa-CD4 cells (Table 3). Entry of BAL andADA into all three C-C CKR-5-expressing cell lines was almost completelyinhibited by the combination of MIP-1α, MIP-1β and RANTES, whereas HxB2entry into LESTR-expressing cells was insensitive to β chemokines (Table3). These results suggest that C-C CKR-5 functions as aβ-chemokine-sensitive second receptor for primary, NSI HIV-1 strains.

The second receptor function of C-C CKR-5 was confirmed in assays ofenv-mediated membrane fusion. When C-C CKR-5 was transiently expressedin COS and HeLa cell lines that permanently expressed human CD4, bothcell lines fused strongly with HeLa cells expressing the JR-FL envelopeglycoproteins, whereas no fusion occurred when control plasmids wereused (data not shown). Expression of LESTR instead of C-C CKR-5 did notpermit either COS-CD4 or HeLa-CD4 cells to fuse with HeLa-JR-FL cells,but did allow fusion between COS-CD4 cells and HeLa-BRU cells (data notshown).

The fusion capacity of β-chemokine receptors was also tested in the RETassay. The expression of C-C CKR-5, but not of C-C CKR-1, -2a, -3 or -4,permitted strong fusion between HeLa-CD4 cells and HeLa-JR-FL cells. Theextent of fusion between HeLa-JR-FL cells and C-C CKR-5-expressingHeLa-CD4 cells was greater than the constitutive level of fusion betweenHeLa-BRU cells and HeLa-CD4 cells (FIG. 4). The fusion-conferringfunction of C-C CKR-5 for primary, NSI HIV-1 strains has therefore beenconfirmed in two independent fusion assays.

EXPERIMENTAL DISCUSSION

Together, the above results establish that MIP-1α, MIP-1β and RANTESinhibit HIV-1 infection at the entry stage, by interfering with thevirus-cell fusion reaction subsequent to CD4 binding. It was also shownthat C-C CKR-5 can serve as a second receptor for entry of primary NSIstrains of HIV-1 into CD4+ T-cells, and that the interaction ofβ-chemokines with C-C CKR-5 inhibits the HIV-1 fusion reaction.

REFERENCES OF THE SECOND SERIES OF EXPERIMENTS

-   1. Levy, J. A., Mackewicz, C. E. & Barker. E. Immunol. Today 17,    217-224 (1996).-   2. Cocchi, F. et al. Science 270, 1811-1815 (1995).-   3. Paxton, W. A. et al. Nat. Med. 2, 412-417 (1996).-   4. Neote, K., DiGregorio, D. Mak, J. Y., Horuk, R., & Schall, T. J.    Cell 72, 415-425 (1993).-   5. Gao, J.-L. et al. J. Exp. Med. 177, 1421-1427 (1993).-   6. Bacon, K. B., Premack, B. A., Gardner, P. & Schall, T. J. Science    269, 1727-1729 (1995).-   7. Raport, C. J. et al. J. Leukoc. Biol. 59.18-23 (1996).-   8. Wells, T. N. C. et al. J. Leukoc. Biol. 59, 53-60 (1996).-   9. Feng, Y., Broder, C. C., Kennedy, P. E. & Berger, E. A. Science    272, 872-877 (1996).-   10. Chen, B. K., Saksela, K., Andino, R. & Baltimore, D. J. Virol.    66, 654-660 (1994).-   11. Connor, R. I., Chen, B. K. Choe, S., & Landau, N. R. Virology    206, 935-944 (1995).-   12. Lusao, P. et al. J. Virol. 69, 3712-3720 (1995).-   13. Charo, l. F. et al. Proc. Natl. Acad. Sci. USA 91, 2752-2756    (1994).-   14. Ben-Baruch, A. et al. J. Biol. Chem. 270, 22123-22128 (1995).-   15. Combadiere, C et al. J. Biol. Chem. 270, 29671-29675 (1995).-   16. Lip, J. P., D'Andrea, A. D., Lodish, H. F. & Baltimore, D.    Nature 343, 762-764 (1990).-   17. Litwin, V. et al. J. Virol. (submitted for publication).-   18. Moore, J. P., Jameson, B. A., Weiss, R. A. & Sattentau, Q. J. in    Viral Fusion Mechanisms (ed Bentz, J.) 233-289 (CRC Press Inc, Boca    Raton, USA, 1993).-   19. Maddon, P. J. et al. Cell 47, 333-348 (1986).-   20. Ashorn, P. A., Berger, E. A. & Moss, B. J. Virol. 64, 2149-2156    (1990).-   21. Clapham, P. R., Blanc, D. & Weiss, R. A. Virology 181, 703-715    (1991).-   22. Samson. M., Labbe, O., Mollereau, C., Vassart, G. &    Parmentier, M. Biochemistry 11, 3362-3367 (1996).-   23. Dragic, T., Charneau. P., Clavel, F. & Alizon. M. J. Virol. 66,    4794-4802 (1992)-   24. Loetscher, M. et al. J. Biol. Chem. 269, 232-237 (1994).-   25. Moore, J. P. & Ho, D. D. AIDS 9 (suppl A), S117-S136 (1995).-   26. Trkola, A. & Moore, J. P. (unpublished data).-   27. Chaudhuri, A., et al. 1994. J. Biol. Chem. 269, 7835-7838    (1994).-   28. Neote, K. Mak, J. Y., Kolakowski Jr., L. F. & Schall, T. J.    Blood 84, 44-52 (1994).-   29. Dragic, T. Picard, L. & Alizon, M. J. Virol. 69, 1013-1018    (1995).-   30. Puri, A., Morris, S. J., Jones, P., Ryan, M. & Blumenthal, R.    Virology 219, 262-267 (1996). 31

Third Series of Experiments

The chemokine SDF-1 (stromal cell-derived factor 1) is the naturalligand for Fusin/CXCR4 and blocks infection by laboratory-adaptedstrains of HIV-1 (Ref. 1 and 2). SDF-1 exists as at least two forms,SDF-1α and SDF-1β based on variable splicing of the SDF-1 gene (Ref. 1and 3) In the RET assay, this chemokine specifically inhibits membranefusion mediated by gp120/gp41 form the laboratory-adapted strainHIV_(LAI) but not by gp120/gp41 from the macrophage-tropic isolateHIV-1_(JR-FL) as shown in FIG. 5.

REFERENCES OF THE THIRD SERIES OF EXPERIMENTS

-   1. Bleul, C. C., et al. (1996) Nature 382:829-833-   2. Oberlin, E., et al. (1996) Nature 382:833-835-   3. Shirozu, M., et al. (1995) Genomics 28:495-500

Fourth Series of Experiments

Direct Binding of HIV-1_(JR-FL) gp120 to CCR5⁺ CD4⁺ Cells

The direct binding of HIV-1_(JR-FL) gp120 to CCR5⁺ CD4⁺ cells has beendemonstrated. In this case, preincubation of the gp120 with sCD4 oranother CD4-based molecule is required, presumably because this resultsin a conformational change in gp120 that exposes a chemokine receptorbinding site. FIG. 6 illustrates the use of flow cytometry to measurethe direct binding of sCD4/gp120 complexes to human CCR5-bearing murineL1.2 cells. Background levels of binding were observed with eitherbiotinylated protein alone, or if gp120 from the laboratory-adaptedstrain HIV-1_(LAI) is used in place of the HIV-1_(JR-FL) gp120 (data notshown).

This assay has been adapted for drug screening purposes to a 96-wellmicroplate format where binding of the sCD4/gp120 complexes toCCR5⁺/CD4⁻ cells is measured using a fluorometric plate reader. Onemethod is as follows:

-   1) Plate out L1.2-CCR5⁺ cells (approx. 500,000/well).-   2) Add inhibitor for 1 hour at room temperature.-   3) Wash and add biotinylated sCD4 (2.5 μg/ml) and biotinylated    HIV-1_(JR-FL) gp120 (5 μg/ml), then incubate for 2 hours at room    temperature.-   4) Wash and incubate with streptavidin-phycoerythrin (100 ng/nl).-   5) Wash and measure the amount of bound gp120/sCD4 using a    fluorometric plate reader exciting at 530 nm and reading emission at    590 nm.

Using this method, inhibition of binding of gp120/sCD4 to CCR5 byCC-chemokines (FIG. 7) and antibodies to CCR5 that block HIV-1 infection(not shown) have been demonstrated.

Inhibition of HIV-1 Envelope-Mediated Membrane Fusion by the Bicyclam,JM3100.

The bicyclam JM3100, obtained from Dr. J. Moore (Aaron Diamond AIDSResearch Center, NY) was tested for ability to inhibit membrane fusionmediated by the envelope glycoproteins of the LAI or JR-FL strains ofHIV-1 using the resonance energy transfer (RET) assay described above.As illustrated in FIG. 7, this molecule specifically and potentlyinhibits fusion mediated by gp120/gp41 from the HIV-1_(LAI) strain, andnot from the HIV-1_(JR-FL) strain. These data suggest that this moleculespecifically inhibits HIV fusion by blocking the interaction betweenHIV-1_(LAI) gp120 and CXCR4.

Fifth Series of Experiments CCR5 Receptor Binding Assay Materials:

-   1. CCR5⁺/L1.2 cell line-   2. L1.2 cell line-   3. JRFL-gp120, biotinylated-   4. sCD4, unconjugated (Intracell, Cat 613101)-   5. 96-well round bottom plate (Corning, cat #25850)-   6. Streptavidin, phycoerythrin conjugated [SA-PE] (Becton Dickinson,    cat #349023)-   7. PBS without Calcium and Magnesium [PBS(−)] (Gibco BRL, cat    #14190)

Method:

-   1. Culture CCR5⁺ and parental L1.2 cells and treat with sodium    butyrate as described (Wu et al., J. Exp. Med 185:1681).-   2. Add cells to 96-well plate (˜3×10⁵ cells/well)-   3. Centrifuge plate and remove supernatant.-   4. Dilute inhibitory compounds as desired in PBS(−)/0.1% NaN₃. Add    40 μl of inhibitory compounds to cells. Add 40 μl of PBS(−)/0.1%    NaN₃ to wells without inhibitory compounds.-   5. Shake plate to suspend cells in solution. Incubate at room    temperature for 1 hour.

6. Prepare an equimolar (˜50 nM) mixture of sCD4 and biotinylated gp120.Add 40 μl of sCD4:biotinylated gp120 complex per well. (Final volume inwell=80 μl). Shake plate to suspend cells in protein solution. Incubateat room temperature for one hour.

-   7. Centrifuge plate and remove supernatant. Add 200 μl of PBS(−)    0.1% NaN₃ per well. Repeat this washing procedure, for a total of    three washes.-   8. Centrifuge plate and remove supernatant. Dilute SA-PE 1:50 in    PBS(−)/0.1% NaN₃ and add 40 μl of diluted reagent to cells. Shake    plate to suspend cells in solution. Incubate at room temperature for    one hour.-   9. Centrifuge plate as above and remove supernatant. Add 200 μl of    PBS(−)/0.1% NaN₃ per well. Repeat this washing procedure for a total    of three washes.-   10. Centrifuge plate as above and remove supernatant. Add 200 μl of    PBS(−)/0.1% NaN₃ per well.-   11. Centrifuge plate and measure the fluorescence. Emission at 590    nm following excitation at 530 nm.-   12. % Inhibition is calculated by using the following formula:

% Inhibition=[Max−Reading]/[Max−Min]

Max=Average of values in wells containing [sCD4: biotinylated gp120w/CCR5+/L1.2 cells, no inhibitor]Min=Average of values in wells containing sCD4:biotinylated gp120 w/L1.2cells, no inhibitor.Reading=Value in specific well

1-60. (canceled)
 61. A CCR5 chemokine receptor antagonist which (a)binds to a CCR5 chemokine receptor on the surface of a CCR5+ CD4+ humancell; (b) inhibits fusion of HIV-1_(JR-FL) with a PM-1 cell; (c) doesnot inhibit fusion of HIV-1_(BUR) with a PM-1 cell; (d) does notactivate an inflammatory response upon binding to the CCR5 chemokinereceptor on the surface of the CCR5+ CD4+ cell; (e) competes withRANTES, MIP-1α and MIP-1β for binding to the CCR5 chemokine receptor onthe surface of the CCR5+, CD4+ cell; and (f) inhibits binding ofHIV-1_(JR-FL) gp120 to the CCR5+, CD4+ cell.